Method of immunizing humans against Salmonella typhi using a Vi-rEPA conjugate vaccine

ABSTRACT

This invention relates to conjugates of the Vi polysaccharide of  S. typhi  with the carrier  Pseudomonas aeruginosa  recombinant exoprotein A (rEPA), and compositions thereof, and to methods of using of these conjugates and/or compositions thereof for eliciting an immunogenic response in humans, including responses which provide protection against, or reduce the severity of,  S. typhi  bacterial infections. The conjugates, and compositions thereof, are useful as vaccines to induce serum antibodies againt  S. typhi  and are useful to prevent and/or treat illnesses caused by  S. typhi.

FIELD OF THE INVENTION

[0001] This invention relates to methods of using conjugates of thecapsular polysaccharide of Salmonella typhi, Vi, bound to the carrierPseudomonas aeruginosa recombinant exoprotein A (rEPA) with a carboxylicacid dihydrazide linker, preferably an adipic acid dihydrazide (ADH)linker, and compositions of these conjugates, for eliciting serumantibody responses in humans, including responses which provideprotection against, or reduce the severity of, S. typhi bacterialinfections. The conjugates, and compositions thereof, are useful asvaccines to induce serum antibodies which are useful to prevent and/ortreat illnesses caused by S. typhi.

BACKGROUND

[0002] In developing countries, typhoid fever is common, serious, andincreasingly difficult to treat because of resistance of the bacillus toantibiotics. [24, 63-66]. For example, more than 80% of Salmonella typhifrom the Mekong Delta region of Vietnam are resistant to chloramphenicoland to ampicillin and even more expensive antibiotics such asciprofloxacin. Typhoid fever has been thought of as a disease of mostlyolder children and young adults. In children less than 5 years of age,typhoid fever was often unrecognized due to atypical clinical symptoms,difficulties in drawing blood and less-than-optimal culture media.[66-69]. Similar to recent findings in other parts of Southeast Asia[70-72], a preliminary survey in 3 communes of the Dong Thap province ofVietnam showed that the annual attack rate of typhoid fever was highestamong children less than 15 years of age: it was 413/100,000 in this agegroup and 358/100,000 for 2 to 4 year-olds. [73].

[0003] Unfortunately, it is unlikely that safe drinking water andfoodstuffs will be available in many developing countries, especially inrural areas, in the near future. [24, 66, 74]. Control of typhoid feverby routine vaccination, especially in countries that endure high endemicrates of typhoid fever, has not been adopted because of the limitationsof the three licensed vaccines (parenteral inactivated cellularvaccines, oral attenuated S. typhi Ty21 a, and parenteral Vipolysaccharide). These vaccines confer only approximately 70% immunityto older children and adults but do not protect young children. [24, 1,30, 75, 76].

[0004] Orally administered attenuated S. typhi Ty21a requires at least 3doses, has a low rate of efficacy in areas with a high rate of typhoidfever and in travelers from developed countries and is not immunogenicin young children. Neither the protective antigens nor thevaccine-induced host immune responses have been identified which hindersimprovement of the Ty21a vaccine.

[0005] Although effective in areas with high rates of typhoid fever,killed whole cell parenteral vaccines elicit a high rate of adversereactions and have not been shown to be effective in young children. In1952, Landy concluded that the protective antigen of cellular vaccinesis the capsular polysaccharide (Vi) of S. typhi.

[0006] In two randomized, double-blinded, vaccine-controlled clinicaltrials, one injection of Vi induced about 70% efficacy in ≧5 year-oldsin the Kathmandu Valley of Nepal and in the Eastern Transvaal region ofthe Republic of South Africa: these regions had a high rate of endemictyphoid (0.4 to 1% per year) [1]. Recently, similar results wereobtained by the Lanzhou Institute of Biologic Products in the People'sRepublic of China [manuscript in preparation]. Vi is easilystandardized. The World Health Organization has published requirementsfor Vi polysaccharide typhoid vaccine and this product is licensed inabout 50 countries including the United States [59,60]. But Vi inducesonly short-lived antibody responses in children two to five years of ageand does not elicit protective levels in children less than two yearsold: in adults, reinjection restores the level of vaccine-inducedanti-Vi but does not elicit a booster response. These age-related andT-independent immunologic properties are similar to most otherpolysaccharide vaccines.

[0007] We proposed that it is the vaccine-induced serum IgG anti-Vi thatconfers immunity. Accordingly, the level of serum IgG anti-Vi shouldpredict the efficacy of Vi vaccine. In order to improve itsimmunogenicity, Vi was conjugated to proteins using SPDP [51, 52, 54,62]. The protein carriers for the SPDP linked conjugates includedcholera toxin (CT), tetanus toxoid (TT), the B subunit of theheat-labile cholera-like enterotoxin (LT-B) of Escherichia coli and therecombinant exoprotein A (rEPA) of Pseudomonas aeruginosa (i.e., thenontoxic recombinant form of exotoxin from Pseudomonas aeruginosa (ETA)cloned into and secreted by E. coli). [Id.). Recently, we employedanother synthesis that treated rEPA with adipic acid dihydrazide (ADH)and bound the hydrazide derivative of rEPA (rEPA-AH) to Vi with1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) [31]. The safetyand immunogenicity of the Vi-rEPA conjugates prepared either withN-succinimidyl-3-(2-pyridyl dithio) propionate (SPDP, Vi-rEPA_(I)) oradipic acid dihydrazide (ADH, Vi-rEPA_(II)) as linkers, were comparedsequentially in adults, 5-14 year-olds and then 2-4 year olds inVietnam. The data set forth in Example 5 herein demonstrate that theresultant conjugate (Vi-rEPA) both enhanced the immunogenicity of andconferred T-cell dependent properties to Vi. Vi-rEPA elicited a boosterresponse in 2 to 4 year-olds with IgG anti-Vi levels approximately 3times higher than those elicited by Vi in 5 to 14 year-olds. None of thevaccinees had a temperature >38.5° C. or swelling >2.5 cm followinginjection. On-the basis of these results, we initiated a double-blindedplacebo-controlled randomized trial to determine the efficacy of Vi-rEPAin 2 to 5 year-old Vietnamese children, an age group for which there isyet no effective typhoid vaccine. The results of that efficacy trial areset forth in Example 6 herein.

BRIEF DESCRIPTION OF THE INVENTION

[0008] It is an object of the invention to provide methods of usingconjugates of the capsular polysaccharide of Salmonella typhi (Vi) boundto the carrier rEPA (as carrier protein) with a carboxylic aciddihydrazide linker, preferably an adipic acid dihydrazide (ADH) linker,and/or compositions thereof, for eliciting an immunogenic response inmammals, including responses which provide protection against, or reducethe severity of, bacterial infections. More particularly, it is anobject of the invention to provide methods of using such conjugates,and/or compositions thereof, to induce serum antibodies against thecapsular polysaccharide of S. typhi, called Vi. The conjugates, andcompositions thereof, are useful as vaccines to induce serum antibodieswhich are useful to prevent typhoid fever.

[0009] It is also an object of the invention to provide antibodies whichimmunoreact with the Vi polysaccharide of S. typhi and/or the rEPAcarrier, that are induced by these conjugates and/or compositionsthereof. Such antibodies may be isolated, or may be provided in the formof serum containing these antibodies.

[0010] It is also an object of the invention to provide a method for thetreatment or prevention of S. typhi infection in a mammal, byadministration of compositions containing the antibodies of theinvention, or serum containing the antibodies of the invention.

[0011] The invention also provides methods and kits for identifying,detecting, and/or diagnosing S. typhi infection or colonization usingthe antibodies which immunoreact with the Vi polysaccharide of S. typhi.The invention also relates to methods and kits for identifying,detecting and/or diagnosing the presence of P. aeruginosa and/or P.aeruginosa exotoxin A (ETA).

[0012] The Vi-rEPA_(II) conjugates of this invention induce a stronginitial IgG antibody response in humans. In this respect, they have asignificant advantage over the Vi-rEPA_(I) conjugates.

DETAILED DESCRIPTION OF THE INVENTION

[0013] The invention provides methods of using conjugates of an S. typhiVi polysaccharide which is covalently bound to the carrier rEPA with adicarboxylic acid dihydrazide linker, preferably an adipic aciddihydrazide linker, and compositions thereof. The present invention alsoencompasses methods of using mixtures such S. typhi-rEPA conjugatesand/or compositions thereof as part of a composition containing otherimmunogens, to form a multivalent vaccine for broad coverage againstvarious pathogens. The S. typhi-rEPA conjugates, and/or compositionsthereof, may also be administered concurrently with other vaccines, suchas the DTP vaccine.

[0014] The invention also provides methods of using such S. typhi-rEPAconjugates, and/or compositions thereof, to induce in mammals, inparticular, humans, the production of antibodies which immunoreact withthe Vi polysaccharide of S. typhi. In the preferred embodiment,antibodies which immunoreact with ETA of P. aeruginosa are alsoproduced. The antibodies which immunoreact with Vi of S. typhi may beuseful for the identification, detection, and/or diagnosis of S. typhicolonization and/or infection. Antibodies against S. typhi may be usefulto prevent and/or treat illnesses caused by S. typhi. Antibodies whichimmunoreact with ETA may be useful to prevent or treat illnesses causedby P. aerugitiosa.

[0015] Pharmaceutical compositions of this invention are capable, uponinjection into a human, of inducing serum antibodies against S. typhi.In general, the exemplified Vi-rEPA conjugate vaccine of this inventionusing ADH as the linker (i.e., Vi-rEPA_(II)) is capable of inducingserum IgG antibody levels which are statistically significantly higherthan those induced by Vi alone or by Vi conjugated to rEPA using SPDP asthe linker (i.e., Vi-rEPA_(I)). The induction by the immunogen, in ≧80%of the immunized population, of a ≧8-fold increase in anti-Vi IgG atfour to six weeks after a proscribed course of vaccination with theimmunogen has been completed, is usually required for an effectivevaccine against typhoid fever.

[0016] Preferably, the method of the invention is capable, uponinjection into an adult human of an amount of Vi-rEPA_(II) vaccinecontaining 25 μg of S. typhi Vi polysaccharide, of inducing in the serumof the human a level of anti-Vi IgG antibody which, when measured sixweeks after the injection, is at least about 48-fold higher than theanti-Vi IgG levels prior to injection.

[0017] Also preferably, the method of the invention is capable, uponinjection into a five- to fourteen-year-old human of an amount ofVi-rEPA_(II) vaccine composition containing 25 μg of S. typhi Vipolysaccharide, of inducing in the serum of the human a level of anti-ViIgG antibody which, when measured six weeks after the injection, is atleast about 252-fold higher than the anti-Vi IgG levels prior toinjection.

[0018] Also preferably, the method of the invention is capable, uponinjection into a two- to four-year-old human of an amount ofVi-rEPA_(II) vaccine composition containing 25 μg of S. typhi Vipolysaccharide, of inducing in the serum of the human a level of anti-ViIgG antibody which, when measured six weeks after the injection, is atleast about 400-fold higher than the anti-Vi IgG levels prior toinjection.

[0019] The Vi-rEPA vaccines of this invention are intended for activeimmunization for prevention of S. typhi infection, and for preparationof immune antibodies. The vaccines of this invention are designed toconfer specific immunity against infection with S. typhi, and to induceantibodies specific to S. typhi Vi and ETA. The S. typhi conjugatevaccine is composed of non-toxic bacterial components, suitable forinfants, children of all ages, and adults.

[0020] The methods of using the Vi-rEPA conjugates of this invention,and/or compositions thereof will be useful in increasing resistance to,preventing, ameliorating, and/or treating S. typhi infection in humans.

[0021] This invention also provides compositions, including but notlimited to, mammalian serum, plasma, and immunoglobulin fractions, whichcontain antibodies which are immunoreactive with S. typhi Vi, and whichpreferably also contain antibodies which are immunoreactive with ETA.These antibodies and antibody compositions may be useful to prevent,treat, or ameliorate infection and disease caused by the microorganism.The invention also provides such antibodies in isolated form.

[0022] High titer anti-Vi sera, or antibodies isolated therefrom, may beused for therapeutic treatment for patients with S. typhi infection.Antibodies elicited by the Vi-rEPA conjugates of this invention may beused for the treatment of established S. typhi infections, and may alsobe useful in providing passive protection to an individual exposed to S.typhi.

[0023] The present invention also provides diagnostic tests and/or kitsfor S. typhi infection and/or colonization, using the conjugates and/orantibodies of the present invention, or compositions thereof.

[0024] The invention is intended to be included in the routineimmunization schedule of infants and children, and in individuals atrisk for S. typhi infection. It is also planned to be used forintervention in epidemics caused by S. typhi. Additionally, it is may beused as a component of a multivalent vaccine for S. typhi and otherpathogens, useful for example for the routine immunization of infants.

[0025] Definitions

[0026] Vi is a linear homopolymer of α(1→4)-D-GalpA, which isN-acetylated at C-2 and O-acetylated at C-3.

[0027] As used herein, the terms “immunoreact” and “immunoreactivity”refer to specific binding between an antigen or antigenicdeterminant-containing molecule and a molecule having an antibodycombining site, such as a whole antibody molecule or a portion thereof.

[0028] As used herein, the term “antibody” refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules. Exemplary antibody molecules are intact immunoglobulinmolecules, substantially intact immunoglobulin molecules and portions ofan immunoglobulin molecule, including those portions known in the art asFab, Fab′, F(ab′)₂ and F(v), as well as chimeric antibody molecules.

[0029] Polymeric Carriers

[0030] Carriers are chosen to increase the immunogenicity of thepolysaccharide and/or to raise antibodies against the carrier which aremedically beneficial. Carriers that fulfill these criteria are wellknown in the art. A polymeric carrier can be a natural or a syntheticmaterial containing one or more functional groups, for example primaryand/or secondary amino groups, azido groups, or carboxyl groups. Carriercan be water soluble or insoluble. The present invention concernsmethods of using Vi conjugates with rEPA as a carrier.

[0031] Methods for Attaching Vi to rEPA

[0032] Methods for binding a polysaccharide to a protein, with orwithout a linking molecule, are well known in the art. See for examplereference [8b], where 3 different methods for conjugating Shigella O-SPto tetanus toxoid are exemplified. See also, reference [31], whichdescribes methods for conjugating S. typhi Vi and adipichydrazide-derivatized protein.

[0033] In the present invention, attachment of the S. typhi Vipolysaccharide to a protein carrier is preferably accomplished by firstcoupling a dicarboxylic acid dihydrazide linker to rEPA, by treatmentwith a peptide coupling agent, preferably a water-soluble carbodiimidesuch as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide,1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide methiodide, or the like,to produce a hydrazide-functionalized carrier protein. Adipic aciddihydrazide is a preferred linker, but conjugates employing otherlinkers, such as the dihydrazides of succinic, suberic, and sebacicacids, are contemplated to be within the scope of the invention. The S.typhi polysaccharide, Vi, is then coupled to thehydrazide-functionalized carrier protein, again preferably with awater-soluble carbodiimide.

[0034] Regardless of the precise method used to prepare the conjugate,after the coupling reactions have been carried out the unbound materialsare removed by routine physicochemical methods, such as for example gelfiltration or ion exchange column chromatography, depending on thematerials to be separated. The final conjugate consists of thepolysaccharide and the carrier bound through a dihydrazide linker.

[0035] Dosage for Vaccination

[0036] The present inoculum contains an effective, immunogenic amount ofa Vi-rEPA polysaccharide-carrier conjugate. The effective amount ofpolysaccharide-carrier conjugate per unit dose sufficient to induce animmune response to S. typhi depends, among other things, on the speciesof mammal inoculated, the body weight of the mammal, and the choseninoculation regimen, as is well known in the art. Inocula typicallycontain polysaccharide-carrier conjugates with concentrations ofpolysaccharide from about 1 micrograms to about 500 micrograms perinoculation (dose), preferably about 3 micrograms to about 50 microgramsper dose, and most preferably about 5 micrograms to 25 micrograms perdose.

[0037] The term “unit dose” as it pertains to the inocula refers tophysically discrete units suitable as unitary dosages for mammals, eachunit containing a predetermined quantity of active material(polysaccharide) calculated to produce the desired immunogenic effect inassociation with the required diluent.

[0038] Inocula are typically prepared in physiologically and/orpharmaceutically tolerable (acceptable) carriers, and are preferablyprepared as solutions in physiologically and/or pharmaceuticallyacceptable diluents such as water, saline, phosphate-buffered saline, orthe like, to form an aqueous pharmaceutical composition. Adjuvants, suchas aluminum hydroxide, may also be included in the compositions.

[0039] The route of inoculation may be intramuscular, subcutaneous orthe like, which results in eliciting antibodies protective against S.typhi. In order to increase the antibody level, a second or booster dosemay be administered approximately 4 to 6 weeks after the initialinjection. Subsequent doses may be administered as indicated herein, oras desired by the practitioner.

[0040] Antibodies

[0041] An antibody of the present invention in one embodiment ischaracterized as comprising antibody molecules that immunoreact with S.typhi Vi.

[0042] An antibody of the present invention is typically produced byimmunizing a mammal with an immunogen or vaccine containing an S. typhiVi-rEPA polysaccharide-protein carrier conjugate to induce, in themammal, antibody molecules having immunospecificity for the immunizingpolysaccharide. Antibody molecules having immunospecificity for theprotein carrier will also be produced. The antibody molecules may becollected from the mammal and, optionally, isolated and purified bymethods known in the art.

[0043] Human or humanized monoclonal antibodies are preferred, includingthose made by phage display technology, by hybridomas, or by mice withhuman immune systems. The antibody molecules of the present inventionmay be polyclonal or monoclonal. Monoclonal antibodies may be producedby methods known in the art. Portions of immunoglobulin molecules, suchas Fabs, may also be produced by methods known in the art.

[0044] The antibody of the present invention may be contained in bloodplasma, serum, hybridoma supernatants and the like. Alternatively, theantibodies of the present invention are isolated to the extent desiredby well known techniques such as, for example, ion chromatography oraffinity chromatography. The antibodies may be purified so as to obtainspecific classes or subclasses of antibody such as IgM, IgG, IgA, IgG₁,IgG₂, IgG₃, IgG₄ and the like. Antibodies of the IgG class are preferredfor purposes of passive protection. The antibodies of the presentinvention have a number of diagnostic and therapeutic uses. Theantibodies can be used as an in vitro diagnostic agents to test for thepresence of S. typhi in biological samples or in meat and meat products,in standard immunoassay protocols. Such assays include, but are notlimited to, agglutination assays, radioimmunoassays, enzyme-linkedimmunosorbent assays, fluorescence assays, Western blots and the like.In one such assay, for example, the biological sample is contacted withfirst antibodies of the present invention, and a labeled second antibodyis used to detect the presence of S. typhi to which the first antibodieshave bound.

[0045] Such assays may be, for example, of direct format (where thelabeled first antibody is reactive with the antigen), an indirect format(where a labeled second antibody is reactive with the first antibody), acompetitive format (such as the addition of a labeled antigen), or asandwich format (where both labeled and unlabelled antibody areutilized), as well as other formats described in the art.

[0046] The antibodies of the present invention are also useful inprevention and treatment of infections and diseases caused by S. typhi.

[0047] In providing the antibodies of the present invention to arecipient mammal, preferably a human, the dosage of administeredantibodies will vary depending upon such factors as the mammal's age,weight, height, sex, general medical condition, previous medical historyand the like.

[0048] In general, it is desirable to provide the recipient with adosage of antibodies which is in the range of from about 1 mg/kg toabout 10 mg/kg body weight of the mammal, although a lower or higherdose may be administered. The antibodies of the present invention areintended to be provided to the recipient subject in an amount sufficientto prevent, or lessen or attenuate the severity, extent or duration ofthe infection by S. typhi. Antibodies which immunoreact with ETA areintended to be provided to the recipient subject in an amount sufficientto prevent, lessen or attenuate the severity, extent or duration of aninfection by ETA producing organisms, such as P. aeruginosa.

[0049] The administration of the agents of the invention may be foreither “prophylactic” or “therapeutic” purpose. When providedprophylactically, the agents are provided in advance of any symptom. Theprophylactic administration of the agent serves to prevent or ameliorateany subsequent infection. When provided therapeutically, the agent isprovided at (or shortly after) the onset of a symptom of infection. Theagent of the present invention may, thus, be provided prior to theanticipated exposure to S. typhi (or other Shiga toxin producingbacteria), so as to attenuate the anticipated severity, duration orextent of an infection and disease symptoms, after exposure or suspectedexposure to these bacteria, or after the actual initiation of aninfection.

[0050] For all therapeutic, prophylactic and diagnostic uses, thepolysaccharide-carrier conjugates of this invention, as well asantibodies and other necessary reagents and appropriate devices andaccessories may be provided in kit form so as to be readily availableand easily used.

[0051] The following examples are exemplary of the present processes andincorporate suitable process parameters for use herein. These parametersmay be varied, however, and the following should not be deemed limiting.

EXAMPLE 1 Materials and Methods

[0052] Clinical protocol. The study was approved by the Ministry ofHealth of Vietnam, the Institutional Review Board of the NationalInstitute of Child Health and Development, NIH and FDA. Informed consentwas obtained from adults or from parents or guardians of vaccinees under18 years old. The site was Cao Lanh District, Dong Thap Province in theMekong Delta region of Vietnam. The vaccines were stored at 4° C. andinjected intramuscularly into the deltoid muscle in 0.5 mL aliquots,containing 25 μg of Vi alone or as a conjugate. Twenty two teachers oradministrative personnel of the Bon Sang Nursery, My-Tho Town, Cao LanhDistrict, received an injection of Vi-rEPA_(II) (BB IND 6990). 157 5 to14 year-olds, recruited from the elementary, middle and high school ofCao Lanh District, received 1 injection of 0.5 mL of either Vi-rEPA_(I)(BB IND 4334), Vi-rEPA_(II) or Vi (Lot K1140, manufactured byPasteur-Mérieux Serums et Vaccins and distributed by ConnaughtLaboratories, U.S. License 384).

[0053] A group of 203 2 to 4 year-olds, recruited from the Bon SangNursery, were randomized to receive either 1 or 2 injections ofVi-rEPA_(I) or of Vi-rEPA_(II) spaced 6 weeks apart. The groups areuneven because some individuals refused the second injection.

[0054] The teachers and the parents were instructed to examine thechildren at 6, 24 and 48 hours following the injection. Children whowere absent on the ensuing 2 days were visited at home by the DistrictHealth medical staff. None of the recipients had erythema >1 cm at theinjection site and none of the recipients had fever for 2 days followingthe injection.

[0055] Blood samples were taken before, and at 6 and 26 weeks afterinjection of the adults and 5-14 year-olds. An additional blood samplewas taken from the 2-4 year-olds 10 weeks after the first injection.

[0056] Reagents: Dialysis tubing: Spectra/por, 45 mm, mwco=3500, 32 mm,mwco=8000, from Spectrum, Houston, Tex.; Biodesign #D102, 15.5,mwco=8000, Carmel, N.Y.; YM10 membrane, 62 mm, mwco=10,000 from Amicon,Beverly, Mass.; Filters: 150 mL unit, C.A., 0.45 μm, from Nalgene,Rochester, N.Y., 25 mm, 0.45 μm, Uniflow, from Schleicher & Schuell,Keen, N H; Chemicals: 2-[N-morpholino]-ethanesulfonic acid [MES], acidform, sodium form, [MES buffer (pH5.6), titrated with MES-Na and MES-H];hydroxylamine, resorcinol, adipic acid dihydrazide [ADH],1-ethyl-3-(3-dimethylaminopropyl) carbodiimide [EDC], thimerosal, fromSigma Chemical Co, St. Louis, Mo; Tris, GIBCO, NY; Sephacryl S-1000,Sephadex G-50, from Pharmacia, Piscataway, N.J.; ammonium sulfate,Mallinckcrodt, Paris, Ky.; Limulus amebocyte lysate (LAL), lot12-56-648, from Associates of Cape Cod Ind. Woods Hole, Mass.; U.S.standard endotoxin (RSE), lot EC-5, from Bureau of Biologics (CBER),FDA, Bethesda, Md.; Goat anti-exotoxin, lot GAE-02A from List BiologicalLab., Inc., Campbell, Calif.; 2,4,6-trinitrobenzene sulfuric acid,Pierce Chemicals, Rockford, Ill.; Coomassie blue reagent, standardbovine serum albumin (BSA) solution (2 mg/mL), from Pierce Chemicals,II.

[0057] Assays. EDC, protein, hydrazide were measured as described [31].Vi content of conjugates was measured by determination of the 0 acetylwith Vi as a standard. Sterility, pyrogenicity, and general safety wasassayed according to the Code of Federal Regulations (CFR) 610.126.

[0058] Vi (Lot 112A). Vi (3.2 μmol O-acetyl/mg, 1.2% nucleic acid,<0.01% protein) was obtained from Pasteur-Merieux, Serums et Vaccins,Lyon, France. This Vi (985 mg) was extracted with cold phenol 10 times.The water phase was dialyzed 4 times against 6 L of pyrogen-free water(PFW), 4° C. and freeze-dried. The final yield of Vi was ˜50%. Theendotoxin content, determined by Limulus Amebocyte Lysate (LAL), was25-50 EU/μg.

[0059] rEPA. Recombinant exoprotein A (rEPA) is a geneticallymanipulated non-toxic, fully antigenic derivative of Pseudomonasaeruginosa exotoxin A (ETA) secreted by the recombinant strain ofEscherichia coli BL21 (IDE3) carrying plasmid pVC45D. Fermentation of E.coli BL21 (IDE3) and purification of rEPA was performed as described[10]. Fractions containing rEPA were pooled, dialyzed againstpyrogen-free saline (PFS), 50 mM sodium phosphate (PBS), pH 7.2, sterilefiltered and ultracentrifuged at 100,000×g for 5 hours at 4° C. Thepellet was discarded and the supernatant (25 mL) was sterile-filtered.The endotoxin content of rEPA was <1 EU/mg. rEPA showed no toxicity inmice at 500 times the lethal dose of ETA.

[0060] Vi-rEPA_(I) (SPDP). Vi-rEPA_(I) (lot 61411) was prepared usingN-succinimidyl-3-(2-pyridyl dithio) propionate (SPDP) as a linker [51,54]. Cystamine (360 mg), dissolved in 20 mL of PFS, was mixed with 120mg of Vi (lot 112A) and the pH brought to 5.0 with 0.1M NaOH in anautotitrator. EDC was added to a final concentration of 0.1M and the pHmaintained at 5.0 for 3 hours by addition of 0.1M HCl. The reactionmixture was dialyzed against PFW at 5° C. and freeze-dried. The SHconcentration was 1.3% w/w.

[0061] SPDP, 14 mg/1.6 mL ethanol, was added to 7 mL of rEPA (10 mg/mL)with stirring for 2 hours at room temperature and overnight at 4° C. Thereaction mixture was passed through a Bio-Gel P-6DG in PBS, 1 mM EDTA,pH 7.2, the void volume concentrated, sterile-filtered, and stored at 4°C. The SPDP/rEPA was 10.6 mol/mol. A single line of precipitation wasformed between rEPA and the rEPA-SPDP derivative (not shown).

[0062] Dithiothreitol (DTT) (37.2 mg) was added to 3 mL of Vi-cystamine(10 mg/mL in PFS, 10 mM sodium phosphate, pH 7.2 (PBS)) for 2 hours atroom temperature. The reaction mixture was passed through Bio-Gel P6DGin PFS. Void volume fractions were sterile-filtered and added to 4.0 mLof rEPA-SPDP (31.5 mg). The reaction mixture was stirred at roomtemperature for 2 hours and passed through a column of S-1000 Sephacrylin PBS, pH 7.2 at 4° C. Fractions were monitored for protein, O-acetyl,and by immunodiffusion. A pool of conjugate-containing fractions (71μg/mL protein and 75 μg/mL Vi) was denoted as Vi-rEPA_(I) Rabbitanti-ETA and burro anti-Vi reacted with an identical precipitation linewith Vi-rEPA_(I) and did not enter 10% PAGE in 1% SDS (not shown).

[0063] Vi-rEPA_(II) (ADH). 0.5M MES buffer, pH 5.6 (4.6 mL), was addedto 24.6 mL of rEPA (300 mg): the resultant pH was 5.7. With stirring,ADH (1.05 g) was added followed by EDC (60.8 mg) and maintained for 1hour at room temperature. The pH was stable at 5.6. The reaction mixturewas dialyzed against PBS at 4° C., centrifuged at 14,500×g for 30minutes at 4° C., and the supernatant passed through a 5×87 cm column ofSephadex G-50 in 0.2 M NaCl, 0.25 mM phosphate, pH 7.0. The void volumefractions were concentrated over a YM-10 membrane at N₂ pressure of 150kPa and sterile-filtered. The ratio of hydrazide/protein of rEPA-AH was0.023 (w/w) or 8.7 (mol/mol). SDS-PAGE (8% acrylamide) showed a similarpattern of rEPA-AH compared to rEPA (not shown). rEPA-AH and rEPA formeda line of identity with goat anti-ETA (not shown).

[0064] Vi, 100 mg of Vi (10 mg/mL PFS) was mixed with 2.4 mL of 0.5 MMES buffer pH 5.6 at room temperature. With mixing, 63 mg of EDC wasadded and after 2 minutes, 100 mg of rEPA-AH (10.1 mg/mL) was addeddropwise. The reaction mixture was brought to 33.3 mL with 11 mL of PFSso that the concentration of Vi and rEPA was 3 mg/mL and 10 mM for EDC.The pH rose gradually from 5.5 to 5.7 within 3 hours of reaction, thenthe pH was brought up to 7.0 with several drops of 1M sodium phosphatebuffer, pH 7.0. The mixture was stored at 4° C. overnight, centrifugedfor 30 minutes at 14,500×g, 10° C. and passed through a 2.5×90 cmSephacryl S-1000 column in phosphate buffered saline, pH 7.0, (PBS,pyrogen-free saline containing 5 mM sodium phosphate pH 7.0, and 0.01%thimerosal). Fractions #23 through 50 were pooled and the bulk ofVi-rEPA contained 200.3 μg Vi/ml and 171.1 μg rEPA/ml. The bulk was4-times diluted with the PBS and the resultant Vi-rEPA_(II) was bottled.The bottled conjugate vaccine contained 48 μg Vi/ml and 43 μg rEPA/ml.

[0065] Immunogenicity in mice and in guinea pigs: Vi or the Viconjugates were diluted to 25 μg/mL in saline and 0.1 mL injectedsubcutaneously three times 2 weeks apart in 6 week-old female generalpurpose mice (10 mice/group). Controls were injected once with saline orthree times with Vi alone. Mice were exsanguinated 7 days after eachinjection and assayed for total Vi antibody by ELISA using a pooledmurine hyperimmune serum, containing 500 μg anti-Vi/mL, as a reference.

[0066] Three vaccines, containing 5 μg of saccharide, were injected intogroups of 4 6-week-old Duncan-Hartley guinea pigs and serum anti-Viassayed by ELISA as described (performed by Pasteur Merieux).

[0067] Double immunodiffusion. Vi, rEPA, rEPA-AH, and the two conjugateswere reacted with burro anti-Vi (B260) and goat anti-EPA and sera frommice after their second injection of conjugates were assayed by doubleimmunodiffusion against 100 μg Vi/mL as described [31].

[0068] ELISA. Total anti-Vi was assayed in murine and in guinea pig seraas described [31]. IgG was extracted from 500 mL of plasma from an adultvaccinated with Vi polysaccharide typhoid vaccine. The anti-Vi contentof this human IgG was assayed by RIA by Pasteur Merieux Serum etVaccins, Lyon, France. Sera were assayed for IgG, IgM and IgA anti-Vi byELISA [31]. Goat anti-human IgG (Jackson Immuno Research Laboratories,Inc) and IgM (Sigma Chemical Company) conjugated to alkaline phosphatasewere used as secondary antibodies. Serum from a typhoid carrier withhigh titer of IgM anti-Vi IgM was assigned a value 100 EU and used as areference for this Ig. The levels of anti-Vi were calculated as apercent of the standard and expressed as the geometric mean and the25-75 centiles for IgG and for IgM anti-Vi. Confirming previous results,the correlation coefficient was r=0.964 between the level of IgG anti-Viassayed by RIA and ELISA and 0.084 for IgM. Antibody levels areexpressed as the geometric mean and the 25th and 75th centiles.

[0069] Data analysis. Comparisons of geometric means were performed bypaired and unpaired t tests when appropriate.

EXAMPLE 2 Composition of the Vaccines

[0070] Because the immunogenicity is related to M_(r) of Vi, we used thehighest molecular weight Vi available for our conjugates. Vi passesthrough Sephacryl S-1000 and the CL-2B Sepharose starting from the voidvolume. SDS-PAGE of the fractions showed a Coomassie Blue-staining bandthat did not move through the gel: there were no bands in the gel.Double immunodiffusion showed a stained circle around the edge of theantigen wells and two precipitation lines that did not cross, onepositive with anti-Vi and the other with anti-ETA sera. Accordingly, wecannot yet distinguish bound (conjugated) from unbound Vi. The specificsfor the sterility and general safety tests in Code of FederalRegulations 610.11 should be met.

[0071] Vi-rEPA_(II) did not give a positive reaction with thesensitivity of the assay at 2.6×10⁻⁵ M carbodiimide. 10% SDS PAGE ofVi-rEPA, or of Vi-rEPA_(II) showed one band at the top of the gel (didnot enter the gel). No bands corresponding to the rEPA-AH or rEPA weredetected. In HPLC profiles of Vi(lot 126A), Vi-rEPA_(I) and ofVi-rEPA_(II), and rEPA on TSK-G6000, Vi eluted as a single broad peak at19.96 minutes and rEPA-AH eluted at 24.27 minutes. Both Vi-rEPA_(I) andVi-rEPA_(II) showed one peak at 16.67 minutes with A₂₈₀. TABLE 1 Vipolysaccharides from Pasteur Mérieux, Serum et Vaccins, Lyon, France.Lot 65332 (Vi-rEPA_(II)) Lot 51706 (Vi-rEPA_(I)) Pasteur Mérieux Vi112AVi104A O-acetyl 3.19 μmoles/mg 2.97 μmoles/mg M_(r) 60.3% <Kd 0.25 60.5%<Kd 0.25 Nucleic acid   1.2%   1.4% Protein <0.1% <0.1% Pyrogen Passes0.01 μg/kg Passes 0.01 μg/kg

EXAMPLE 3 Immunogenicity in Mice

[0072] After one injection, mice immunized with either conjugate hadhigher levels of anti-Vi than Vi alone (13,4, 112.5 vs 5.78 p=0.01). Incontrast to Vi, both conjugates elicited a booster response after thesecond injection (79.5 vs 12.5, 109 vs 13.4, p=0.01): Vi-rEPA_(II)elicited higher levels of anti-Vi than Vi-rEPA, (109 vs 79.5, p=0.05).See Table 2. Sera from the mice after the second injection of eitherconjugate precipitated with Vi in double immunodiffusion (not shown).TABLE 2 Geometric mean serum anti-Vi (μg/mL) elicited in mice bysubcantaneous injection of Vi, Vi-rEPA_(I) and Vi-rEPA_(II) Immunogen n=1st injection 2nd injection Saline 5 0.05 Not done Vi, Lot 104A 10 1.37Not done Vi, Lot 112A 10 3.72 Not done Vi-rEPA_(I) 10 8.82 59.2Vi-rEPA_(II*) 10 12.6 79.5

[0073] Numbers for Vi Lot 1 04A and for related conjugate Vi-rEPA_(I)are reported in Ref. 54 (see general purpose mice experiment). Resultsare in μig anti-Vi/mL (measured by RIA).

[0074] Vi-rEPA_(II) and related Lot of Vi 112A (used for making thisconjugate) were made and tested in animals years after Vi-rEPA_(I) wasmade and tested in mice, guinea pigs and adults. Immunogenicity ofVi-rEPA_(II) was tested by ELISA, not RIA, but because results areexpressed in μg anti-Vi-mL using the same standard serum, the numbers inTable 2 are comparable.

EXAMPLE 4 Immunogenicity in Guinea Pigs

[0075] As reported, Vi did not elicit anti-Vi in guinea pigs after twoinjections [31]. Neither conjugate induced anti-V_(I) after the firstinjection and both conjugates elicited anti-Vi after the secondinjection, but only Vi-rEPA_(II) elicited a statistically significantrise of the GM anti-Vi level after the third injection. See Table 3.TABLE 3 Geometric mean serum IgG anti-Vi (ELISA units) elicited inguinea pigs (n = 4) by injection of Vi, Vi-rEPA_(I) and Vi-rEPA_(II)Immunogen 1st injection 2nd injection 3rd injection Saline <10 <10 Notdone Vi, Lot 104A <2 <2 <2 Vi, Lot 112A <10 <10 <10 Vi-rEPA_(I) <31 384167 Vi-rEPA_(II*) <10 60 54

[0076] Vi-rEPA, is reported in Ref. 54. Results from the guinea pigexperiment with Vi-rEPA_(I) and Vi Lot 104 cannot be compared withVi-rEPA_(II), because they are expressed in arbitrary ELISA unitsreferring to different standard sera.

EXAMPLE 5 Clinical Reactions

[0077] None of the volunteers had fever following either the first orsecond injection. Local reactions were confined to mild pain in a smallfraction of the vaccinees at any age.

[0078] Anti-Vi in adults (Table 4). in the present study, onlyVi-rEPA_(II) was evaluated in adults. All volunteers had significantlyhigher pre-existing anti-Vi than those of the 5-14 year-olds (9.62 vs0.44, 0.42, 0.61, p=0.0001). Six weeks after injection, all vaccineesresponded with >4 rise in anti-Vi of the 3 Ig classes: 48-fold rise ofIgG (465 vs 9.62, p=0.0001), 5-fold rise of IgM (19.0 vs 4.76, p=0.0001)and a 43-fold rise of IgA (8.85 vs 0.20, p=0.0001). The IgG anti-Vi fellto 119 (3.9-fold decline) at 26 weeks, but this level was 12.4-foldhigher than the pre-vaccination levels (119 vs 9.62, p=0.0001).Similarly, at the 26 weeks interval, IgM and IgA anti-Vi declined butwere significantly higher than the pre-immune values (p<0.01). TABLE 4Serum anti-Vi (μg/mL) in adults (n = 22) elicited by one injection ofVi-rEPA_(II) Pre-injection 6 weeks 26 weeks IgG: Geometric mean 9.62 465119 25-75 centiles  5.0-20.8 293-894 52.8-277  465, 119 vs 9.62, p =0.0001; 465 vs 119, p = 0.0001 IgM: Geometric mean 4.76 19.0 9.34 25-75centiles 2.68-7.48 6.27-36.2 4.78-18.2 19.0, 9.34 vs 4.76, p < 0.0.01;19.0 vs 9.34, NS IgA: Geometric mean 0.20 8.85 4.99 25-75 centiles0.10-0.30 1.92-18.2 1.22-10.7 8.85, 4.99 vs 0.20, p = 0.0001; 8.85 vs4.99, p < 0.0001

[0079] Vi-rEPA_(I) was evaluated in adults in a previous study using 15ug polysaccharide per injection, as reported in reference 54.

[0080] Anti-Vi in 5-14 year-olds (Table 5). On a random basis, the 5-14year-olds were injected once with Vi or 1 of the 2 conjugates. All 4groups had similar levels of pre-injection anti-Vi that weresignificantly lower than those of adults (vide supra).Serum  anti-Vi  of  5  to  14  year-olds  injected  with  Vi, Vi − r  EPA_(I)  or  Vi − rEPA_(II)  Geometric  mean  ELISA  Units  (25-75  th  centiles)  $\begin{matrix}\quad & \quad & {Vi} & \quad & {{Vi} - {rEPA}_{I}} & \quad & {{Vi} - {rEPA}_{II}} \\\quad & \quad & \quad & \quad & \quad & \quad & \quad \\{{IgG}\text{:}{Pre}\text{-}} & \quad & {0.44( {0.28 - 0.59} )} & \quad & {0.42( {0.24 - 0.53} )} & \quad & {0.67( {0.24 - 1.81} )} \\{6\quad {wks}} & \quad & {18.9( {7.84 - 44.1} )} & \quad & {22.8( {7.86 - 58.9} )} & \quad & {169.0( {80.8 - 290} )} \\{26\quad {wks}} & \quad & {13.4( {6.01 - 29.4} )} & \quad & {10.8( {3.64 - 28.8} )} & \quad & {30.0( {14.1 - 45.5} )}\end{matrix}$   30.0  vs  13.4, 10.8; p < 0.001 $\begin{matrix}{{IgM}\text{:}{Pre}\text{-}} & \quad & {6.47( {4.02 - 9.90} )} & \quad & {6.75( {4.16 - 10.2} )} & \quad & {5.79( {3.33 - 8.25} )} \\{6\quad {wks}} & \quad & {25.2( {17.4 - 40.3} )} & \quad & {48.0( {21.0 - 81.1} )} & \quad & {92.1( {51.5 - 154} )} \\{26\quad {wks}} & \quad & {12.3( {6.64 - 21.2} )} & \quad & {26.2( {13.0 - 49.0} )} & \quad & {31.3( {17.9 - 56.7} )}\end{matrix}$  31.3  vs  26.2  NS; 31.3  vs  12.3p = 0.0001;  31.3, 26.2  vs  12.3p = 0.0002$\begin{matrix}{{IgA}\text{:}{Pre}\text{-}} & \quad & {0.05( {0.03 - 0.07} )} & \quad & {0.03( {0.02 - 0.04} )} & \quad & {0.05( {0.02 - 0.10} )} \\{6\quad {wks}} & \quad & {2.64( {0.81 - 7.59} )} & \quad & {1.99( {0.73 - 5.13} )} & \quad & {16.5( {9.19 - 43.5} )} \\{26\quad {wks}} & \quad & {2.04( {0.81 - 6.72} )} & \quad & {0.99( {0.35 - 2.77} )} & \quad & {4.99( {3.34 - 28.9} )}\end{matrix}$   4.99  vs  2.04, NS;  4.99  vs  0.99, p = 0.02

[0081] All three vaccines elicited significant rises of anti-Vi of the 3isotypes at 6 and at 26 weeks over the pre-immune levels. Vi-rEPA_(II)elicited higher levels of anti-Vi at all intervals than Vi-rEPA_(I) andVi.

[0082] IgG anti-Vi. At 6 weeks, all responded with >4-fold rises ofanti-Vi: 43-fold for Vi, 54-fold for Vi-rEPA_(I) and 252-fold forVi-rEPA_(II). Vi-rEPA_(II) elicited higher levels of anti-Vi thanVi-rEPA_(I) or Vi (169 vs 22.8, 18.9 p=0.0001). Twenty six weeks later,the IgG anti-Vi of all groups declined but remained >4 fold higher thanthe pre-immune levels: Vi-rEPA_(II)>Vi>Vi-rEPA_(I) (30.0 vs 13.4,10.8,p=0.0001). Of interest, is that similar levels of IgG anti-Vi wereelicited by Vi-rEPA_(I) and Vi at both 6 and 26 weeks followingvaccination.

[0083] IgM anti-Vi. Pre-immune levels of the three groups were similar.At the six weeks interval, all the vaccines elicited significant risesof anti-Vi (25.2 vs 6.47, 48.0 vs 6.75, 92.1 vs 5.79; p=0.0001, for Vi,Vi-rEPA_(I), for Vi-rEPA_(II), respectively). Vi-rEPA, induced higheranti-Vi than Vi alone at both post vaccination intervals (p<0.0002). At26 weeks, the GM IgM anti-Vi of the three groups were higher than thepre-immune levels: the levels in the recipients of the conjugates werehigher than that of Vi (31.3,26.2 vs 12.2; p<0.01).

[0084] IgA anti-Vi. The pre-immune levels of the three groups weresimilar and almost at the level of detection. Vi-rEPA_(II) elicited thehighest IgA anti-Vi of the 3 vaccines (16.5 vs 1.99, 2.64; p<0.002). Thelevels of the 3 groups declined at 26 weeks but this order of IgAanti-Vi was retained at 26 weeks (4.99 for Vi-rEPA_(II) vs Vi-rEPA_(I),2.04; NS; 4.99 vs 0.99, p=0.02). The 26 week level elicited byVi-rEPA_(II) (4.99) was higher than the 6 week level in the groupsreceiving Vi (2.04) and Vi-rEPA_(I) (1.99).

[0085] One vs 2 injections of Vi conjugates in 2 to 4 year-olds (Table6) Vi was not administered to the 2-4 year-olds. On a random basis, 2-4year-olds were administered 1 or 2 injections of Vi-rEPA, orVi-rEPA_(II) 6 weeks apart: blood was taken before each injection and 4and 26 weeks after the second injection. The pre-immune levels of the 4groups were similar and slightly lower than those of the 5-14 year-olds.Six weeks after the first injection, all responded with >4 fold rise ofanti-Vi of each Ig class and there was no significant difference foreach conjugate between the groups destined to receive 1 or 2 injections.TABLE 6 Serum anti-Vi of 2-4 year-olds injected 1 or 2 times 6 weeksapart with Vi-rEPA_(I) or Vi-rEPA_(II) (˜50/group) Geometric mean μgAb/mL (25-75th centiles) Vi-rEPA_(I) Vi-rEPA_(II) 1 inj. 2 inj. 1 inj. 2inj. IgG Pre 0.32 (0.23-0.40) 0.33 (0.23-0.43) 0.19 (0.10-0.27) 0.18(0.11-0.23)  6 wk 30.2 (15.2-53.5) 28.9 (18.0-53.0) 77.2 (41.3-165) 69.9 (36.5-126)  10 wk 21.4 (10.9-39.8) 83.0 (46.3-185)  54.3(34.5-165)  95.4 (60.0-126)  26 wk 5.50 (2.90-9.80) 12.8 (9.66-25.1)20.4 (9.82-40.9) 30.6 (22.4-51.6) 77.2, 69.9 vs 30.2, 28.9, p = 0.0001;95.4, 83.0 vs 18.9, 22.8 (Table 5), p = 0.0001; 95.4 vs 83.0, NS; 30.6vs 20.4, NS; 30.6 vs 12.8, 5.50, p = 0.0001 IgM: Pre- 4.72 (2.67-7.90)5.00 (3.06-7.48) 3.61 (2.50-4.80) 3.93 (2.84-5.18)  6 wk 37.7(24.1-55.2) 41.8 (26.0-62.7) 47.5 (27.8-81.5) 39.8 (22.9-57.5) 10 wk35.7 (20.3-65.2) 82.5 (51.2-155)  34.8 (20.1-58.6) 31.8 (19.3-48.6) 26wk 19.5 (12.2-29.4) 36.2 (21.8-62.1) 20.1 (13.1-32.3) 19.5 (12.8-30.6)IgA: Pre- 0.02 (0.01-0.02) 0.02 (0.01-0.02) 0.02 (0.01-0.02) 0.02(0.01-0.02)  6 wk 1.76 (1.30-2.54) 1.32 (0.71-3.34) 6.23 (2.79-18.1)5.68 (2.22-12.9) 10 wk 1.48 (1.03-2.68) 2.00 (0.74-3.69) 4.21(1.86-9.90) 4.99 (2.24-11.8) 26 wk 0.70 (0.50-1.12) 0.85 (0.50-2.02)3.00 (1.37-8.49) 2.62 (1.09-7.29) 3.00, 2.62 vs 0.70, 0.85 p < 0.02;6.23 vs 5.68, 4.21 vs 4.99, 3.00 vs 2.62, NS

[0086] On a random basis, 2-4 year-olds were injected 1 or 2 times 6weeks apart with Vi-rEPA_(I) or Vi-rEPA_(II). Blood was drawn beforeeach injection and 4 and 20 weeks after the 2nd injection. NS=notstatistically significant.

[0087] IgG anti-Vi: Vi-rEPA_(II) elicited higher levels of anti-Vi thanVi-rEPA_(I) (77.2, 69.9 vs 30.2, 28.9, p=0.0001). The levels of anti-Viin the 2-4 year-olds receiving Vi-rEPA_(II) were higher than thoseadministered Vi alone in the 5-14 year-olds (77.2,69.9 vs 18.9;p=0.0001). Four weeks after the second injection, both conjugateselicited a rise in anti-Vi (2.87-fold for Vi-rEPA_(I) and 1.36-fold forVi-rEPA_(II): levels elicited by 2 injections of Vi-rEPA_(II) were onlyslightly higher than those by Vi-rEPA_(I) (95.4 vs 83.0, NS). The secondinjection of both conjugates elicited higher levels of anti-Vi than oneinjection of the Vi or Vi-rEPA_(I) in the 5-14 year-olds (95.4, 83.0 vs18.9, 22.8; p=0.0001). At 26 weeks, IgG anti-Vi of the recipients of 2injections of Vi-rEPA_(II) were the highest (30.6 vs 20.4, 12.8, 5.50),similar to that of the 5-14 year-olds receiving the same conjugate buthigher than the recipients of Vi alone in that age group (30.6 vs 13.4,p=0.0001). Serum IgG anti-Vi in the recipients of 2 injections ofVi-rEPA_(II) at 26 weeks were slightly different for the 3 (20.7, n=19)and 4 year-olds (31.4, n=12) compared to the 2 year-olds (20.7, n=6) butthese differences were not statistically significant.

[0088] IgM anti-Vi. IgM anti-Vi levels in all groups were similar andslightly lower than the 5-14 year-olds. One injection of eitherconjugate elicited about an 8-fold increase: there were no significantdifferences between the two conjugates. Unexpectedly, reinjection ofVi-rEPA_(I) (82.5 vs 41.8; p=0.0003), but not Vi-rEPA_(II) (31.8 vs39.8; NS), elicited a booster response. At 26 weeks, the group thatreceived Vi-rEPA_(I) had the highest level; the levels were similar forthe others (36.2 vs 19,5, 20.1, 19.5; NS)

[0089] IgA anti-Vi. Both conjugates elicited a significant rise of IgAanti-Vi: Vi-rEPA_(II) higher than Vi-rEPA_(I) (6.23, 5.68 vs 1.87, 1.36;p=0.02). Reinjection of Vi-rEPA_(I) only elicited a slight rise of IgAanti-Vi (2.00 vs 1.32, NS). The levels of all groups declined at 26weeks although all were significantly higher than those of thepre-immune sera: the recipients of 1 or 2 injections of Vi-rEPA_(II)were similar and higher than those of the groups that receivedVi-rEPA_(I) (3.00, 2.62 vs 0.70, 0.85; p<0.02).

[0090] Anti-rEPA. Anti Vi-rEPA elicited higher levels of IgM anti-Vithan Vi alone in 5-14 year olds (data not shown).

Discussion of Initial Clinical Trials

[0091] As shown for other polysaccharides, such as Haemophilusinfluenzae type b, the immunogenicity of Vi is improved by covalentlybinding it to a protein. Previously, we reported the enhancedimmunogenicity of conjugates compared to Vi, similar to Vi-rEPA_(I), inadults [54].

[0092] Since reinjection of Vi and other polysaccharide-proteinconjugates in, older children or in adults does not elicit a boosterresponse, only 1 injection of Vi conjugates and Vi were compared in 5 to14 year-olds. Unexpectedly, at 6 and at 26 weeks after vaccination, IgGanti-Vi levels elicited by Vi-rEPA_(I) and by Vi were similar at 6 (22.8vs 18.9) and at 26 weeks (10.8 vs 13.4). Vi-rEPA_(I), however, elicitedhigher levels of IgM anti-Vi than Vi at 6 (48.0 vs 25.2) and at 26 weeks(26.2 vs 12.3). Vi-rEPA_(II) elicited higher levels of IgG, IgM and IgAanti-Vi at all intervals in the 5 to 14 year-olds than Vi-rEPA_(I) andVi.

[0093] In the 2-4 year-olds at 26 weeks, IgG anti-Vi levels elicited byVi-rEPA_(II) were higher than those elicited by Vi-rEPA_(I) after 1(20.4 vs 5.50, p=0.01) and 2 injections (30.6 vs 20.4), but these latterdifferences were not statistically significant. Two injections ofVi-rEPA_(II), elicited higher levels than Vi in the 5-14 year-olds and,therefore, it can be predicted that this Vi conjugate will elicitgreater than 70% efficacy when injected 2 times in individuals ≧2 yearsof age. In fact, in a clinical efficacy trial in 2-5 year-olds inVietnam, where typhoid fever is endemic, this Vi conjugate (i.e.,Vi-rEPA_(II)) demonstrated greater than 91% efficacy in preventingtyphoid fever. (See Example 6, hereinbelow).

[0094] There is evidence that a critical (protective) level of serum IgGanti-Vi is sufficient to confer immunity to typhoid fever. In passiveimmunization experiments with serum taken mice and humans injected wihcellular vaccines, IgG anti-Vi accounted for the protection againstchallenge with S. typhi. By analogy to H. influenzae type b and othercapsulated pathogens it is antibodies of the IgG isotype, not IgM orIgA, that exude onto the epithelial surface and account for most of theserum anti-Vi in the intestine. We suggest that measurement of serum IgGanti-Vi will be essential and sufficient to standardize Vi conjugatevaccines.

[0095] We are yet unable to demonstrate by physico-chemical orimmunologic methods whether there is some free Vi (unbound to protein)in our new conjugate. Vi is molecularly polydisperse material thatcannot be 100% effectively separated from Vi conjugates on the availablegel filtration media or polyacrylamide gels. Similarly, doubleimmunodiffusion with antibodies to the Vi and to the carrier protein(rEPA) does not yield a precipitin line of identity. For the present,our only method for identifying that the Vi and protein are covalentlybound is to demonstrate the increased immunogenicity of our conjugate inmice and in guinea pigs compared to mixtures of Vi and the adipichydrazide-derivatized protein. [See, e.g., ref. 31].

Summary of Initial Clinical Trials

[0096] A Vi conjugate, prepared by treatment of an adipic hydrazidederivative of rEPA and Vi with EDC (Vi-rEPA_(II)), was shown to be safeand more immunogenic in mice, guinea pigs and in young children andadults than a similar construct made with SPDP. Vi-rEPA_(II) elicited abooster response in 2-4 year-olds that results in levels of IgG anti-Visignificantly higher than those achieved by Vi alone in 5 to 14year-olds. This new Vi conjugate is safe and can be expected to confer ahigh degree and long-lived immunity against typhoid fever in children aswell as in adults.

[0097] One injection of Vi-rEPA_(II) into adults (n=22) elicited 465ELISA U/mL (48-fold geometric mean rise) of IgG anti-Vi at 6 weeks thatfell to 119 after 26 weeks: similar patterns were observed for IgM andIgA anti-Vi. In 5-14 year-olds (˜50/group), one injection of Vi eliciteda 43-fold rise of IgG anti-Vi, Vi-rEPA_(I) a 54-fold rise (54 vs 43, NS)and Vi-rEPA_(II) a 252-fold rise (252 vs 54,43 p=0.0001). At 26 weeks,Vi-EPA_(II) elicited 30.0 units of IgG anti-Vi that was higher than thatinduced by Vi-rEPA_(I) and Vi (10.8 vs 13.4, NS); all were higher thanpre-immune levels (p=0.0001). Vi-rEPA_(II) elicited the highest IgM andIgA anti-Vi at 6 weeks and at 26 weeks.

[0098] One or two injections of Vi-rEPA_(I) and Vi-rEPA_(II) wereevaluated in the 2-4 year-olds (˜50 group). After 6 weeks there was a406-fold rise of IgG anti-Vi in recipients of Vi-rEPA_(II) and 94-foldrise in recipients of Vi-rEPA_(I) (p=0.0001). Four weeks after a secondinjection, recipients of Vi-rEPA_(I) and Vi-rEPA_(II) had a rise of IgGanti-Vi: 83.0 from 28.9 and 95.4 from 69.9, respectively. At 26 weeks,the IgG anti-Vi levels of all vaccinees were higher than the pre-immunelevels (p=0.0001). IgG anti-Vi levels elicited by 2 injections werehigher than those with only 1 injection (30.6 vs 20.4 for Vi-rEPA_(II)and 12.8 vs 5.50 for Vi-rEPA_(I)): IgG anti-Vi levels elicited by 2injections of Vi-rEPA_(II) were higher than those elicited by Vi in the5-14 year-olds (30.6 vs 13.4, p=0.01). In all three age groups,Vi-rEPA_(II) was more immunogenic than Vi-rEPA_(I). Similar values wereobtained for IgM and IgA anti-Vi. One injection of Vi-rEPA_(II) shouldconfer a higher degree of immunity to typhoid fever than Vi inindividuals ≧5 years: 2 injections should confer comparable immunity in2 to 4 year-olds to that in individuals ≧5 years of age.

EXAMPLE 6 Efficacy Trial Methods

[0099] Study Design

[0100] The Study Protocol was approved by the Institutional ReviewBoards of the National Institute of Child Health and Human Development(NICHD) (Number OH-98-CH-N002) and that of the Ministry of Health,Vietnam, by the Center for Biologics Evaluation and Research, Food andDrug Administration (BB-IND 6990) and was assigned a Single ProjectAssurance (Number S-11089-07) by the Office for Human ResearchProtection, U.S. Department of Health and Human Services.

[0101] In November, 1997, a census of 16 communes in the Cao LanhDistrict, Dong Thap Province in the Mekong Delta of Vietnam, identified14,285 2 to 5 year-olds. Almost all of the households relied upon riverand rain as a source of water. Cao Lanh District is served by TheProvincial Hospital and each commune (5,000 to 20,000 population) has ahealth center with a physician, assistant physicians, nurses, andapproximately 20 community health workers. Approximately 95% of thepopulation is engaged in agriculture.

[0102] Enrollment

[0103] Informed consent for 13,776 2 to 5-year-olds (96.4%) was obtainedfrom the parents/guardians during group meetings and/or house visitsconducted by the health workers. Excluded were children who requiredon-going medical care. Children with parental consent received a healthcard with an unique seven digit identification number.

[0104] Vaccines

[0105] Vi-rEPA was prepared and characterized as described.²⁷⁻²⁹ TheVi-rEPA conjugate contained 22.5 μg Vi (Aventis Pasteur, Lot 126A, Lyon,France) and 22 μg of rEPA in 0.5 mL of saline, 0.01% thimerosal: itsappearance from that of the placebo was indistinguishable. The 5-dosevials, containing 2.8 mL each of Vi-rEPA or of placebo, were labeledwith a number from 0 through 9 on a random basis and packaged 10 perbox. The code, kept by the Pharmacy Department, Clinical Center, NIH andby the Chairman, Safety Monitoring Committee, Ho Chi Minh City, Vietnam,was opened on Jun. 23, 2000.

[0106] Vaccination Protocol

[0107] Two rounds of vaccination were held in 1998. The first was fromFebruary 21 through March 9 and the second from April 4 through April20. Vaccinations were conducted by 64 teams. Children were injected 2times approximately 6 weeks apart with 0.5 mL from a vial with a numberidentical to the last digit of his/her identification number. Prior toinjection, the children were examined by the health staff and theiraxillary temperatures taken. Those with no fever (<37.5° C.) wereinjected into the left deltoid and the vial number recorded. Thechildren were observed for 20 minutes, their temperature taken and theinjection site inspected for redness and swelling at 6, 24 and 48 hoursby a community health worker.

[0108] Case Detection

[0109] The diagnosis of typhoid fever was made only on the demonstrationof S. typhi from a blood culture. Recipients were visited weekly andtheir history and axillary temperature taken by community healthworkers. Children with fever (≧37.5° C.) for at least 3 days werereferred to the health station and 6 mL of blood drawn: 5 mL weredelivered to Difco BACTO Blood Culture bottle, #0936-37-6 and 1 mL forserology. Blood cultures were maintained at 37° C. and the clotted bloodat refrigerator temperature: the samples were brought to The ProvincialHospital on the same day. Cultures were checked at 1, 2 and 7 days andS. typhi identified by established biochemical and serologic assays atthe Microbiology Laboratory, The Provincial Hospital and by Dr. VeeGill, Clinical Microbiology, NIH. All isolates were also verified for Viby the antiserum agar technique at the NICHD: there was no discrepancyamong the three laboratories. [81].

[0110] No additional cases of typhoid fever in the participants werefound in the bacteriologic records of The Provincial Hospital and theDistrict Hospital, adjacent to the south part of the Cao Lanh District.The study was closed on May 31, 2000, 27 months after the firstinjection.

[0111] Immunogenicity and Persistence of Vi-rEPA-Induced IgG Anti-Vi

[0112] Paired sera were obtained from 76 participants before the firstand 4 weeks after the second injection. To evaluate the duration ofvaccine-induced anti-Vi, a 2 mL blood sample was taken at random from 4vaccinees of each commune each month after the second injection. SerumIgG Vi antibodies were assayed by ELISA as described and expressed inELISA units (EU). [79].

[0113] Statistical Analysis

[0114] Vaccine efficacy is expressed as proportionate reduction of casesof typhoid fever: 1−(attack rate in recipients of Vi-rEPA/attack rate inplacebo group)×100%. Confidence limits were calculated by the method ofMiettinen and Nurminen. [82]. Chi Square, or where appropriate, theFisher exact test were used for comparison of categorical variables.Logarithms of the antibody concentrations were used in all calculations.Antibody levels were expressed as the geometric mean (G.M.) with 25thand 75th centiles. Comparisons of G.M. were performed with the unpairedt test or paired t test.

Results

[0115] Characteristics of the Vaccinees (Table 7)

[0116] A total of 12,008 children received at least 1 injection: 11,091(92.4%) received 2 injections (5,525 Vi-rEPA and 5,566 placebo), 771(6.4%) received I injection (388 Vi-rEPA and 383 placebo) and 146 (1.2%)were injected from a vial with an incorrect code (78 Vi-rEPA and 68placebo). The sex, age at vaccination, household composition and size,and interval between the two injections were similar between the vaccinegroups. The interval between the 2 injections ranged from 28 to 57 days(median 42). TABLE 7 Characteristics of participants in the Vi-rEPA andplacebo groups Characteristics Vi-rEPA (n = 5,991) Placebo (n = 6,017)Male 3,033 50.6% 3,120 51.9% Received two 5,525 5,566 injectionsReceived only one 388 383 injection Received incorrect 78 68 injectionNumber of households 5,076 5,082 Adults >18 years/ 2.89 2.94 householdChildren ≦18 years/ 2.74 2.76 household Age at vaccination 2 years 1,33822.3% 1,356 22.5% 3 years 1,422 23.7% 1,418 23.6% 4 years 1,628 27.2%1,597 26.5% 5 years 1,603 26.8% 1,646 27.4% Days between the twoinjections Median (range) 42.0 (29-57) 42.0 (28-56)

[0117] Adverse Reactions (Table 8)

[0118] Serious side reactions were not noted. The reactions from allintervals after injection (20 minutes, 6, 24, 48 hours) were combined.After the first injection, 113 participants had a temperature of ≧37.5°C.: 81 recipients of Vi-rEPA vs 32 recipients of placebo (p<0.001). Ofrecipients with a temperature of ≧39.0° C., 17 were injected withVi-rEPA and 5 with placebo (p=0.01). None of the recipients had ≧5 cm oferythema or swelling.

[0119] After the second injection, a temperature of ≧37.5° C. wasrecorded for 109 recipients of Vi-rEPA and for 25 recipients of placebo(p<0.001). One recipient of Vi-rEPA and one of placebo had a temperature≧39.0° C. Swelling ≧5 cm was noted in 20 recipients of Vi-rEPA comparedto I in the placebo group (p<0.001). Erythema ≧5 cm without swelling wasnoted in 2 recipients of Vi-rEPA and none in the placebo group.

[0120] None of these reactions persisted for more than 48 hours afterinjection. TABLE 8 Local reactions and fever following the first andsecond injections of Vi-rEPA and the placebo (saline). First injectionSecond injection Vi-rEPA Placebo V-rEPA Placebo n= n= p= n= n= p = Fever≧37.5° C. 81 1.36% 32 0.54% <0.001 109 1.93% 25 0.44% <0.001 Fever≧39.0° C. 17 0.29% 5 0.08% 0.01 1 0.02% 1 0.02 NS Swelling ≧5 cm 0 0 200.35% 1 0.02% <0.001 Erythema ≧5 cm 0 0 2 0.04% 0 NS

[0121] Following injection, the axillary temperatures of the childrenwere taken and the injection sites inspected for redness and swelling at6, 24 and 48 hours by a community health worker. The reactions at theintervals were combined.

[0122] Efficacy (Table 9)

[0123] During the surveillance period (Mar. 8, 1998 through May 31,2000), 2,335 blood cultures were obtained: 1,121 from recipients ofVi-rEPA and 1,214 from recipients of the placebo. S. typhi was isolatedfrom 61 recipients representing all communes (34 males and 27 females).The number of typhoid fever cases in each commune ranged from 1 to 9(median 4).

[0124] Among children who received 2 injections from vials withcorrectly allocated vials (fully vaccinated), there were 4 cases oftyphoid fever in the Vi-rEPA group and in the placebo group 47 (efficacy91.5%, 95% C.I. 77.1-96.6). One additional patient received twoinjections of placebo from vials with an incorrectly allocated code.

[0125] Of children that received only 1 injection, there was 1 case oftyphoid fever 42 days after injection of Vi-rEPA and 8 cases throughoutthe study period in the placebo group. Combining all the cases oftyphoid fever, there were 5 in the Vi-rEPA group and 56 in the placebogroup (efficacy 91.0%, 95% C.I. 78.6-96.5). The cases in both theVi-rEPA and placebo groups were distributed evenly among the two to fiveyear-olds. There were two cases during the first year and three casesduring the second year after vaccination in the Vi-rEPA group.

[0126] Among all cases of typhoid fever, 21 (34%) were hospitalized foran average of 13 days (median 12, range 7 to 24): all were in theplacebo group (0/5 vs 21/56, NS).

[0127] A total of 339 vaccinees (165 Vi-rEPA and 174 placebo) were lostto follow-up (2.8%): 308 moved out of the study area, 2 withdrew fromthe study and 29 died (12 Vi-rEPA vs 17 placebo) due to drowning (19),dengue fever (3), pneumonia (2), Stevens Johnson Syndrome (2), and 1each of burns, foreign body in airway, and leukemia. No death wasattributed to the vaccination or to typhoid fever.

[0128] There were four isolates of Salmonella paratyphi A: 1 receivedVi-rEPA and 3 received placebo. TABLE 9 Efficacy of Vi-rEPA conjugatevaccine against typhoid fever in fully vaccinated children and in thosethat received only 1 injection: Dong Thap Province, Vietnam, Mar. 8,1998 through May 31, 2000 Group Vi-rEPA Placebo Efficacy (95% C.I.)Fully vaccinated (n=) 5,525 5,566 Typhoid fever (n=) 4 47 91.5%(77.1-96.6) Attack rate (cases/10³) 0.72 8.44 All participants* (n =)5,991 6,017 Typhoid fever (n=) 5 56 91.0% (78.6-96.5) Attack rate(case/10³) 0.84 9.31

[0129] Characteristics of All Typhoid Fever Patients Vi-rEPA PlaceboMale 5 (100%) 29 (52%) NS Female 0 (0%)  27 (48%) Age at vaccination(years) 2 2 (40%)  16 (29%) NS 3 1 (20%)  17 (13%) 4 0 (0%)  16 (29%) 52 (40%)  17 (30%) Time period of isolation of S. typhi March1998-February 1999 2 (40%)  33 (59%) NS (12 months) March 1999-May 20003 (60%)  23 (41%) (15 months)

[0130] Among those who received only 1 injection, 1 case of typhoidfever occurred 42 days after injection of Vi-rEPA and 8 cases from 25days to 1 year after injection of placebo.

[0131] Serum Levels of IgG Vi Antibodies Before the First and 4 WeeksAfter the Second Injection (Table 10)

[0132] A total of 76 paired sera were available from participants bledbefore their first and 4 weeks after their second injection. There wasno significant difference among the pre-first injection levels of IgGanti-Vi in the two groups and the post-4 week immunization level inrecipients of the placebo. Vi-rEPA, in contrast, elicited approximatelya 660-fold increase in IgG anti-Vi (p<0.0001): 100% of the recipients ofVi-rEPA had ≧10-fold rise. TABLE 10 Serum anti-Vi IgG of participantsbefore their first injection and four weeks after their second injectionby age at vaccination ELISA units [Geometric mean (25^(th) to 75^(th)centiles)] Vaccine 4 weeks post Group n= Pre-1^(st) injection 2^(nd)injection Vi-rEPA (all) 36 0.11 (0.06-0.18) 72.9 (50.7-124) 2-3 years 130.10 (0.06-0.12) 69.0 (56.0-186) 4-5 years 23 0.13 (0.07-0.26) 75.2(46.9-124) Placebo (all) 40 0.15 (0.06-0.19) 0.27 (0.08-0.55) 2-3 years13 0.13 (0.07-0.20) 0.13 (0.07-0.14) 4-5 years 23 0.16 (0.07-0.20) 0.40(0.09-1.39)

[0133] 72.9 vs 0.27, 0.11; p=0.0001, 75.2 vs 72.9 vs 69.0, NS

[0134] Serum IgG anti-Vi was assayed as described in Example 5 herein.[79]. Study participants were injected with the coded vials two timessix weeks apart. Blood samples were taken prior to their first injectionand 4 weeks after the second injection. 100% of the recipients ofVi-rEPA had ≧10-fold rise of their serum IgG anti-Vi.

[0135] Persistence of Vi-rEPA-Induced Anti-Vi (Table 11)

[0136] Blood samples, from 4 randomly selected recipients of eachcommune, were taken each month: only those at 4 intervals (0, 6 months,1 and 2 years) are shown in Table 5. The pre-immunization IgG anti-Vilevels of the Vi-rEPA and placebo groups were similar. At 6 monthsfollowing the second injection, the IgG anti-Vi levels in the recipientsof Vi-rEPA were 22.5 EU or 35-fold higher than those of the placebogroup (p<0.001). At 2 years, Vi-rEPA-induced IgG anti-Vi levelsdecreased approximately 2.1 fold from those at 6 months to 10.7 EU: thislevel was 18.8-fold higher than the controls (10.7 vs 0.57, p<0.001).

[0137] There was a slight rise of the placebo group from 0.15 EU to 0.57(NS).

[0138] A tendency for an age-related immunogenicity and persistence ofIgG anti-Vi was observed when the vaccinees were stratified by age intotwo to three year-olds and four to five-year-olds. The four to fiveyear-olds had a higher level of IgG anti-Vi at all threepost-immunization intervals. The decline of IgG anti-Vi levels from 6months to 2 years was less in the 4 to 5-year-olds (1.4-fold) comparedto the 2 to 3-year-olds (2.4-fold) (NS). TABLE 11 Persistence of serumIgG anti-Vi in fully vaccinated children by age at vaccinationstratified into 2-3 and 4-5 year-old age groups ELISA units (Geometricmean, 25^(th) to 75^(th) centiles) Vaccine Interval after immunizationgroup Pre- 6 months 1 year 2 years Vi-rEPA (all) 0.12 (0.06-0.20) 22.5(13.8-47.3) 18.7 (10.3-32.6) 10.7 (6.4-24.8)  2-3 years 0.12 (0.05-0.23)18.6 (13.1-47.3) 14.3 (7.1-18.6)   7.6 (6.2-17.4)  4-5 years 0.13(0.07-0.20) 25.1 (13.8-49.0) 21.4 (14.0-37.5) 18.4 (8.2-41.4)  Placebo(all) 0.15 (0.06-0.25) 0.65 (0.28-1.03) 0.31 (0.17-0.52) 0.57(0.15-2.50) 2-3 years 0.10 (0.05-0.18) 0.50 (0.27-0.51) 0.30 (0.15-0.38)0.70 (0.26-1.13) 4-5 years 0.20 (0.07-0.59) 1.00 (0.33-1.72) 0.30(0.17-0.59) 0.50 (0.15-2.50)

[0139] 10.7 vs 0.57, p<0.001; 18.4 vs 10.7 vs 7.6, NS

[0140] A blood sample was taken at random from 4 participants at each ofthe 16 communes every month for 24 months. Only the G.M. serum IgGanti-levels at the 0, 6 months and 1 and 2 years intervals are shownabove.

[0141] The 3rd year antibody persistent levels for the phase II Vi-rEPAconjugate study are as follows: ELISA units [Geometric mean (25^(th) to75^(th) centiles)] 2-4 yr Adults(n = 20) 5-14 yr old (n = 8) old (1 inj,n = 28) (2 inj, n = 28) 92.64 4.80 4.83 4.56

[0142] Levels of Serum IgG Anti-Vi from Patients.

[0143] Only 3 sera from the 4 blood samples drawn for culture from thefully vaccinated patients (recipients of Vi-rEPA) were available: theirlevels were 4.76, 14.6 and 40.3 EU. There were 37 sera from typhoidfever cases in the placebo group whose IgG anti-Vi levels ranged from0.05 EU to 3.7 in 36 sera, G.M. 0.41 EU: one patient in the placebogroup had 85.8 EU.

Discussion of Efficacy Trial

[0144] Two novel observations from this trial merit emphasis. One isthat the efficacy for Vi-rEPA (approximately 91.5%) is the highestreported for any typhoid vaccine. The other is the demonstration of theefficacy of a typhoid vaccine, for the first time, in young children. Ahigh degree of efficacy was predicted by the immunogenicity of Vi-rEPAin 2-4 year-olds compared to that of Vi in adults and in 5-14 year-olds.[Example 5 herein, 79]. Since it is the level of serum IgG anti-Viinduced by a Vi-based vaccine that determines its efficacy, we predictthat Vi-rEPA will be at least as effective in older children and adultsas it was shown to be for the 2-5 year-olds. [47]. We are conductingpassive surveillance of the participants for the next two years toevaluate the duration of protection and persistence of IgG anti-Viinduced by Vi-rEPA.

[0145] Vi-rEPA was safe. We subtracted the number of reactions in theplacebo group from that of the Vi-rEPA recipients for each category.After the first injection 0.9% of the vaccinees had a temperature ≧37.5°C. and 0.2% had ≧39.0° C. attributable to Vi-rEPA.

[0146] After the second injection, swelling ≧5 cm at the injection sitewas observed in 20 of Vi-rEPA recipients compared to 1 in the placebogroup. Fever attributable to Vi-rEPA occurred in 1.4% who hadtemperature ≧37.5° C. but none had ≧39.0° C. This degree of safety wasobserved in our Phase 2 trial of Vi-rEPA and could be predicted by theobservation that Vi-rEPA met the Requirements of the World HealthOrganization for Vi polysaccharide vaccine. [Example 5 herein, 79, 80].

[0147] Although the numbers are not statistically significant there is asuggestion that patients who received 1 or 2 injections of Vi-rEPA andwho developed typhoid fever (n=5) had a milder course than the controls(n=56) because 21 of latter (37.5%) were hospitalized compared to noneof the former. We speculate that the higher levels of IgG anti-Vi intyphoid fever cases vaccinated with Vi-rEPA compared to those injectedwith placebo could explain this milder course of disease. The higherlevels of IgG anti-Vi in these patients is likely mediated by a T-cellprimed B-cell population induced by the Vi-rEPA.

[0148] There was a comparable number of blood cultures taken fromrecipients of Vi-rEPA (n=1211) and of placebo (n=1214). S. typhiisolated from the placebo group (56/1214) represents 4.6% children with≧3 days of fever. But the diagnosis of typhoid fever by the results of asingle blood culture is not efficient. [13, 83, 84]. It is estimatedthat only approximately 50% of typhoid fever cases are identified bythis technique compared to culture of the bone marrow. In addition, theyield of S. typhi from blood cultures is related to the duration offever. For example, in our epidemiologic investigation of typhoid feverin this study area, we found that the yield of S. typhi from bloodcultures from children after 3 days of fever was 4.6% compared to 17%from those with ≧7 days of fever. [73]

[0149] The unusual structure, molecular size, and physico-chemicalproperties of Vi contributed to the laborious development of Vi-rEPA.[31, 47, 51, 52, 53, 54, 79]. Both the safety and immunogenicity ofVi-rEPA in this trial were comparable to those of a similar productevaluated in Phase 1 and 2 studies in Vietnam indicating consistency inthe production of this new vaccine. [Example 5 herein, 79].Vi-rEPA-induced levels of IgG anti-Vi declined approximately 2-fold inthe ensuing 2 years but there was no change in the efficacy of thevaccine. Based upon the level in the 2-3 year-olds at the 2 yearinterval after vaccination with Vi-rEPA, we propose that IgG anti-Vi ofno more than 8 EU is the minimal protective level. [85]

[0150] Our objective is to provide a safe and effective typhoid fevervaccine for routine vaccination of infants. Accordingly, we plan anevaluation of the immunogenicity of Vi-rEPA when administered with DTP.Should Vi-rEPA elicit levels of anti-Vi in infants comparable to thosein the 2 to 5 year-olds, this new typhoid vaccine could be administeredas part of the WHO Expanded Program on Immunization. [24]. In addition,Vi-rEPA can be predicted to be a highly effective vaccine for themilitary and travelers to areas with high rates of typhoid fever. [65].

Summary of Efficacy Trial

[0151] Background Typhoid fever is common and serious in developingcountries. Licensed vaccines for typhoid fever confer only about 70%immunity, do not protect young children and are not used for routinevaccination. A newly developed conjugate of Salmonella typhi Vipolysaccharide, bound to a mutant nontoxic Pseudomonas aeruginosaexotoxin A (rEPA), demonstrated enhanced immunogenicity in adults, 5-14and 2-4 year-olds.

[0152] Methods In a double-blinded, placebo-controlled, randomizedtrial, the safety and efficacy of Vi-rEPA was evaluated in 2-5 year-oldsof 16 communes in Dong Thap Province, Mekong Delta, Vietnam.Participants (11,091) received 2 injections 6 weeks apart of Vi-rEPA orplacebo (saline). Typhoid fever was diagnosed by isolation of S. typhifrom blood cultures after ≧3 days of fever (>37.5° C.) was identified byactive surveillance for 27 months. Efficacy was estimated by comparingthe attack rate of typhoid fever in recipients of Vi-rEPA to thecontrols.

[0153] Results S. typhi was isolated from 4 fully vaccinated recipientsof Vi-rEPA compared to 47 of placebo (efficacy 91.5%, 95% C.I.77.1-96.6). Cases were distributed evenly by age and throughout thestudy. Among recipients of only 1 injection, there was 1 case in theVi-rEPA group and 8 in the placebo group. Serious side reactions werenot observed.

[0154] Conclusions Vi-rEPA was safe, immunogenic and elicited >90%efficacy in 2 to 5 year-olds. The high level of serum IgG Vi antibodiesand of efficacy in 2-5 year-olds indicate that Vi-rEPA will be at leastas protective in older individuals including travelers and the military.

REFERENCES

[0155] 1. Acharya, I. L., Lowe, C. U., Thapa, R., Gurubacharya, V. L.,Shrestha, M. B., Bryla, D. A., Cramton, T., Trolifors, B., Cadoz, M.,Schulz, D., Armand, J., Schneerson, R., and Robbins, J. B. 1987.Prevention of typhoid fever in Nepal with the Vi capsular polysaccharideof Salmonella typhi: A preliminary report one year after immunization.N. Engl. J. Med. 317:1101-1104

[0156] 2. Blaser, M. J., R. Newman. 1982. A review of humansalmonellosis: I. Infective dose. Rev. Infect. Dis. 4:1096-1106

[0157] 3. Bodhidatta, L., D. N. Taylor, U. Thisyakorn, and P.Echeverria. 1987. Control of typhoid fever in Bangkok, Thailand, byannual immunization of school children with parenteral typhoid vaccine.Rev. Infect. Dis.9:841-845

[0158] 4. Bradford M M. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing the principleof protein-dye binding. Anal Biochem 1976; 72:248-254

[0159] 5. Brugier, J-C, A. Barra, D. Schulz, J-L. Preud'homme. 1993.Isotypes of human vaccinal antibodies to the Vi capsular polsyaccharideof Salmonella typhi. Int. J. Clin. Lab. Res. 23:38-41

[0160] 6. Cameron D M, R J Collier. 1987. Exotoxin A of Pseudomonasaeruginosa: substitution of glutamic acid 553 with aspartic aciddrastically reduces toxicity and enzymatic activity. J. Bacteriol.169:4967-4971

[0161] 7. Fass R, van de Walle M, Shiloach A, Joslyn A, Kaufman J,Shiloach J. Use of high density cultures of Escherichia coli for highlevel production of recombinant Pseudomonas aeruginosa exotoxin A. ApplMicrobiol Biotechnol 1991; 36:65-69

[0162] 8. (a) Chu, C-Y, R. Schneerson, J. B. Robbins, S. C. Rastogi.1983. Further studies on immunogenicity of Haemophilus influenzae type band pneumococcal 6A polysaccharide-protein conjugates. Infect Immun40:245-256; (b) C. Chu, et al. 1991. Infect. Immun., 59:4450-4458.

[0163] 9. Fattom, A., Vann, W. F., Szu, S. C., Sutton, A., Li, X.,Bryla, D., Schiffman, G., Robbins, J. B., and Schneerson, R. 1988.Synthesis and physiochemical and immunological characterization ofpneumococcus type 12F polysaccharide-diphtheria toxoid conjugates.Infect. Immun. 56:2292-2298

[0164] 10. Fattom, A., J. Shiloach, D. A. Bryla, D. Fitzgerald, 1.Pastan, W. W. Karakawa, J. B. Robbins, R. Sclineerson. 1992. Comparativeimmunogenicity of conjugates composed of the Staphylococcus aureus type8 capsular polysaccharide bound to carrier proteins by adipic aciddihydrazide or N-succinimidyl-3-(2-pyridyldithio)propionate. Infect.Immun. 60:584-589

[0165] 11. Field R. Biochem J 1971; 124:581-590

[0166] 12. Gaines, S., J. A. Currie, and J. G. Tully. 1960. Productionof incomplete Vi antibody in mice. Proc. Soc. Exp. Biol. Med.104:602-605.

[0167] 13. Gilman, R. H., M. Terminel, M. M. Levine, P.Hernandez-Mendoza, R. B. Hornick. 1975. Relative efficacy of blood,urine, rectal swab, bone-marrow, and rose-spot cultures for recovery ofSalmonella typhi in typhoid fever. Lancet. i:1211-1213

[0168] 14. Gotschlich, E. C., M. Rey, W. R. Sanborn, R. Triau and B.Cvjetanovic. 1972. The immunological responses observed in field studiesin Africa with Group A meningococcal vaccines. Progress inImmunobiological Stand. 129:485-491.

[0169] 15. World Health Organization Expert Committee on BiologicalStandardization. 1977. Technical Report Series, 610. WHO, Geneva,Switzerland.

[0170] 16. Jacobson, B S and K R Fairman. 1980. A colorimetric assay forcarbodiimides commonly used in peptide synthesis and carboxyl groupmodification. Anal Biochem 106, 114.

[0171] 17. Johansson, H. J., C. Jagersten, and J. Shiloach. 1996. Largescale recovery and purification of periplasmic protein from E. coliusing explanded bed adsorption chromatograhy followed by new ionexchange media. J. Biotech. 48:9-14.

[0172] 18. Kim, Y-R., J-H. Yoo, J-K. Hur, J. H. Kang, W-S. Shin, M-W.Kang. 1995. Immunogenicity of Vi capsular polysaccharide vaccineevaluated for 3 years in Korea. K Korean Med. Sci. 10:314-317

[0173] 19. Heidelberger, M., M. M. DiLapi, M. Siegel and A. W. Walter.1950. Persistance of antibodies in human subjects injected withpneumococcal polysaccharides. J. Immunol. 65:535-541

[0174] 20. Hestrin S. The reaction of acetylcholine and other carboxylicacid derivatives with hydroxylamine, and its analytical application. JBiol Chem 1949; 180:249-2261

[0175] 21. Hien, T. T., D. B. Bethell, N. T. T. Hoa, J. Wain, T. S.Diep, L. T. Phi, B. M. Cuong, N. M. Duong, P. T. Thanh, A. L. Walsh, N.P. J. Day and N. J. White. 1995. Short course of Ofloxacin for treatmentof multidrug-resistant typhoid. Clin. Infect. Dis. 20:915-923

[0176] 22. Hochstein, H. D. 1990. Role of the FDA in regulating theLimulus amoebocyte lysate tests, p. 38-49. In R. B. Prior (ed). Clinicalapplication of the Limulus amoebocyte lysate test. CRC Press, Inc., BocaRaton, Fla.

[0177] 23. Hornick, R. B., S. E. Greisman, T. E. Woodward, H. L. DuPont,A. T. Dawkins, and M. J. Snyder. 1970. Typhoid fever: pathogenesis andimmunologic control.(second of two parts) N. Eng. J. Med. 283:739-746

[0178] 24. Ivanoff, B., M. M. Levine and P. H. Lambert. 1994.Vaccination against typhoid fever: Present status. Bull. W.H.O.72:957-971

[0179] 25. Jacobson B S, Fairman K R. A colorimetric assay forcarbodiumides commonly used in peptide synthesis and carboxyl groupmodification. Anal Biochem 1980; 106:114-117

[0180] 26. Kawata, Y. 1970. A study of the molecular types ofimmunoglobulin. II. Mouse protection study of Vi antibody againsttyphoid infection. Acta Medicine Univ. Kioto. 40:284-290

[0181] 27. Keitel W A, Bond N L, Zahradnik J M, Cramton T A, Robbins JB. Clinical and serological responses following primary and boosterimmunization with Salmonella typhi Vi capsular polysaccharide vaccines.Vaccine 1994; 12:195-199

[0182] 28. Kim, Y. R., J. H. Yoo, J. K. Hur, J. H. Kang, W. S. Shin, M.W. Kang. 1995. Immunogenicity of Vi capsular polysaccharide vaccineevaluated for three years in Korea. J. Korean Med. Sci. 10:314-317

[0183] 29. Klugman, K. P., Gilbertson, I. T., Koornhof, H. J., Robbins,J. B., Schneerson, R., Schulz, D., Cadoz, M., Armand, J., and VaccineAdvisory Committee. 1987. Protective activity of Vi capsularpolysaccharide vaccine against typhoid fever. Lancet ii: 1165-1169

[0184] 30. Klugman, K. P., H. J. Koornhof, J. B. Robbins, N. M. LeCam.1996. Immunogenicity, efficacy and serological correlate of protectionof Salmonella typhi Vi capsular polysaccharide vaccine three years afterimmunization. Vaccine 14:435-438

[0185] 31. Kossaczka, Z., Bystricky, S., Bryla, D. A., Shiloach, J.,Robbins, J. B., and Szu, S. C. 1997. Synthesis and imnmnunologicalproperties of Vi and di-o-acetyl pectin conjugates with adipic aciddihydrazide as the linker. Infect. Immun. 65:2088-2093

[0186] 32. Kumate, J., J. L. Penaloza and A. Llausas. 1974. La fiebretifoidea en el primer ano de la vida. Bol. Med. Hosp. Infant Mex.31:925-932

[0187] 33. Landy, M. 1954. Studies in Vi antigen. VI. immunization ofhuman beings with purified Vi antigen. Amer. J. Hyg. 60:52-62.

[0188] 34. Landy, M., A. G. Johnson and M. E. Webster. 1961. Studies onVi antigen. VIII. Role of acetyl in antigenic activity. Amer. J. Hyg.73:55-65

[0189] 35. Landy, M., S. Gaines, J. R. Seal, and J. E. Whiteside. 1954.Antibody responses of man to three types of antityphoid immunizingagents: Heat-phenol fluid vaccine, acetone-dehydrated vaccine andisolated Vi and O antigens. Amer. J. Publ. Hlth. 44:1572-1579.

[0190] 36. Levin, D. M., K-H Wong, H. Y. Reynolds, A. Sutton, and R. S.Northrup. 1975. Vi antigen fron Salmonella typhosa and immunity againsttyphoid fever. II. Safety and antigenicity in humans. Infect. Immun.12:1290-1294

[0191] 37. Lukac M, G B Pier, R J Collier. Toxoid of Pseudomonasaeruginosa exotoxin A generated by deletion of an active-site residue.Infect Immun 1988; 56:3095-3098

[0192] 38. Murphy, J. R., L. Grez, L. Schlesinger, C. Ferreccio, S.Baqar, C. Munoz, S. S. Wasserman, G. Losonsky, J. G. Olson, M. M.Levine. Immunogenicity of Salmonella typhi Ty21a vaccine for youngchildren. Infect. Immun. 59:4291-4298

[0193] 39. Monsigny M, et al. Colorimetric determination of neutralsugars by a resorcinol sulfuric acid micromethod. Anal Biochem 1988;175:525-530

[0194] 40. Mirza, N. B., I. A. Wamola, B. A. Estambale, E. Mbithi, M.Pollet. 1995. Typhim Vi vaccine against typhoid fever: a clinical trialin Kenya. East Afr. 72:162-164

[0195] 41. Muschel, L. H. and H. P. Treffers. 1956. Quantitative studieson the bactericidal actions of serum and complement. III. Observationson sera obtained after T.A.B. vaccination or during tyhoid fever. J.Immunlol. 76:20-27.

[0196] 42. Nguyen, T. A., H. B. Khiem, N. T. Dung. 1993. Le fievretyphoid au sud Viet-Nam, 1990-1993. Bull. Soc. Path. Ex. 86:476-478

[0197] 43. Pittman, M. and H. J. Bohner. 1966. Laboratory assays ofdifferent types of field trial typhoid vaccines and relationship toefficacy in man. J. Bacteriol. 91:1713-1723.

[0198] 44. Qadri, A. 1997. Identification of specific recognitionmolecules on murine mononuclear phagocytes and B lymphocytes for Vicapsular polysaccharide: modulation of MHC class II expression onstimulation with the polysaccharide. Immunology 92:146-152

[0199] 45. Robbins, J. D., and Robbins, J. B. 1984. Re-examination ofthe immunopathogenic role of the capsular polysaccharide (Vi antigen) ofSalmonella typhi. J. Infect. Dis. 150:436-449.

[0200] 46. Robbins, J. B., and Schneerson, R.: Polysaccharide-proteinconjugates: A new generation of vaccines. J. Infect. Dis. 161:821-832,1990.

[0201] 47. Robbins, J. B., Schneerson, R., and Szu, S. C.: Perspective:Hypothesis: Serum IgG antibody is sufficient to confer protectionagainst infectious diseases by inactivating the inoculum. J. Infect.Dis. 171:1387-1398, 1995.

[0202] 48. Rowe, B., L. R. Ward and E. J. Threlfall. 1990. Spread ofmultiresistant Salmonella typhi. Lancet 336:1065

[0203] 49. Saha, S. K., S. K. Saha. 1994. Antibiotic resistance ofSalmonella typhi in Bangladesh. J. Antimicrob. Chemother. 33:190-191

[0204] 50. Schneerson, R., Barrera, O., Sutton, A., and Robbins, J. B.1980. Preparation, characterization and immunogenicity of Haemophilusinfluenzae type b polysaccharide-protein conjugates. J. Exp. Med.152:361-376.

[0205] 51. Szu, S. C., Stone, A. L., Robbins, J. D., Schneerson, R., andRobbins, J. B.: Vi capsular polysaccharide-protein conjugates forprevention of typhoid fever. J. Exp. Med. 166:1510-1524, 1987.

[0206] 52. Szu, S. C., Li, X., Schneerson, R., Vickers, J., and Robbins,J. B.: Comparative immunogenicities of Vi polysaccharide-7proteinconjugates composed of cholera toxin or its B-subunit as a carrier boundto high or lower molecular weight Vi. Infect. Immun. 57:3823-3827, 1989.

[0207] 53. Szu, S. C., X. Li, A. L. Stone, J. B. Robbins, J. B. 1991.Relation between the structure and immunologic properties of the Vicapsular polysaccharide. Infect. Immun. 59:4555-4561.

[0208] 54. Szu, S. C., D. N. Taylor, A. C. Trofa, J. D. Clements, J.Shiloach, J. C. Sadoff, D. A. Bryla, J. B. Robbins. 1994. Laboratory andpreliminary clinical characterization of Vi capsularpolysaccharide-protein conjugate vaccines. Infect. Immun. 62:4440-4444.

[0209] 55. Am, N. T., Khiem, H. B., Dung, N. T. 1993. La fièvre typhoïdeau Sud du Viet-Nam, 1990-1993. Bull. Soc. Path. Ex. 86:476-478

[0210] 56. Wong, K. H., J. C. Feeley, and M. Pittman. 1972. Effect of aVi-degrading enzyme on potency of typhoid vaccines in mice. J. Infect.Dis. 125:360-366

[0211] 57. Wong, K. H. and J. Feeley. 1972. Isolation of Vi antigen anda simple method for its measurement. App. Microbiol. 24:628-633

[0212] 58. Wong, K. H., J. C. Feeley, M. Pittman, and M. E. Forlines.1974. Adhesion of Vi antigen and toxicity in typhoid vaccinesinactivated by acetone or by heat and phenol. J. Infect. Dis.129:501-506

[0213] 59. World Health Organization Expert Committee on BiologicalStandardization. 1977. Technical Report Series, 610. WHO, Geneva,Switzerland.

[0214] 60. World Health Organization Expert Committee on BiologicStandardization. Technical Report Series 840, 43rd Ed Geneva,Switzerland. Requirements on Vi polysaccharide for typhoid. 1993: 14-32.

[0215] 61. Yoshihiro J, V. K. Chaudhary, T. Kondo, S. Adhya, D. J.Fitzgerald, I. Pastan. 1988. Mutational analysis of domain I ofPseudmonas exotoxin. J. Biol. Chem. 263:13203-13207

[0216] 62. U.S. Pat. No. 5,204,098, Polysaccharide Protein Conjugates,Szu et al.

[0217] 63. Bhutta Z A, Naqvi S H, Razzaq R A, and Farooqui B J.Multidrug-resistant typhoid in children: presentation and clinicalfeature. Rev Infect Dis 1991; 13:832-6.

[0218] 64. Wain J, Nguyen T T H, Nguyen T C, et al. Quinolone-resistantSalmonella typhi in Vietnam: Molecular basis of resistance and clinicalresponse to treatment. Clin Infect Dis 1997; 25:1404-10.

[0219] 65. Mermin J H, Townes J M, Gerber M, Dolan N, Miontz E D, TauxeR V. Typhoid fever in the United States, 1985-1994: changing risks ofinternational travel and increasing antimicrobial resistance. ArchIntern Med 1998; 158:633-8.

[0220] 66. Mermin J H, Villar R, Carpenter J, el al. A massive epidemicof multi-resistant typhoid fever in Tajikistan associated withconsumption of municipal water. J Infect Dis 1999; 179:1416-22.

[0221] 67. Rao PT, Rao KVK. Typhoid fever in children. Indian J Pediatr1959; 26:258-64.

[0222] 68. Scragg J, Rubidge C, and Wallace H L. Typhoid fever inAfrican and Indian children in Durban. Arch Dis Child 1969; 44: 18-28

[0223] 69. Galloway H, Clark N S and Blackhall M. Paediatric aspects ofthe Aberdeen typhoid outbreak. Arch Dis Child 1986; 41:63-8.

[0224] 70. Thisyakom U, Mansuwan P, Taylor D N. Typhoid and paratyphoidfever in 192 hospitalized children in Thailand. Am J Dis Child 1987; 141:862-85.

[0225] 71. Simanjuntak C H, Paleologo F P, Punjabi N H, et al. Oralimmunization against typhoid fever in Indonesia with Ty21a vaccine.Lancet 1991; 338:1055-9.

[0226] 72. Sinha A, Sazawal S, Kumar R, et al. Typhoid fever in chldrenaged less than 5 years. Lancet 1999; 354:734-7.

[0227] 73. Lin F Y, Ho V A, Bay P V, et al. The epidemiology of typhoidfever in the Dong Thap Province, Mekong delta region of Vietnam. Am JTrop Med Hyg. Amer J Trop Med Hyg, in press

[0228] 74. Tarr P E, Kuppin S L, Jones T C, Ivanoff B, Aparain P G,Heymann D C. Consideration regarding mass vaccination against typhoidfever as an adjunct to sanitation and public health measures: potentialuse in an epidemic in Tajikistan. Amer J Trop Med 1999: 61:163-170.

[0229] 75. Levine M M, Ferreccio C. Abrego P, Martin O S, Ortiz E, CryzS. Duration of efficacy pf Ty21a, attentuated Salmonella typhi live oralvaccine. Vaccine 1999; 17:Suppl 2:S22-7.

[0230] 76. Centers for Disease Control and Prevention. Typhoidimmunization: recommendations of the Advisory Committee on ImmunizationPractices. MMWR Morb Mortal Wkly Rep 1994; 31:1-7.

[0231] 77. Felix A, Pitt R M. A new antigen of B. typhosus. Lancet 1934;186-91.

[0232] 78. Robbins J B, Schneerson R, Anderson P, Smith D H. Preventionof systemic infections, especially meningitis, caused by Haemophilusinfluenzae type b: Impact on public health and implications for otherpolysaccharide-based vaccines. JAMA 1996; 276:1181-5.

[0233] 79. Kossaczka Z, Lin F Y, Ho A V, et al. Safety andimmunogenicity of Vi conjugate vaccines for typhoid fever in adults, 5to 14 year-olds, and 2 to 4 year-olds in Vietnam. Infect Immun 1999;67:5806-10.

[0234] 80. World Health Organization. Annex 1: Requirements for Vipolysaccharide typhoid vaccine (Requirements for Biological SubstancesNo. 48). WHO Technical Report Series 1994; No. 840 15-29.

[0235] 81. Nolan C M, LaBorde E A, Howell R T, Robbins J B.Identification of Salmonella typhi in faecal specimens by an antiserumagar method. J Med Microbiol 1980; 13:373-7.

[0236] 82. Miettinen O, Nuriminen M. Comparative analysis of two rates.Statistics in Medicine, 1985; 4:213-26.

[0237] 83. Escamilla J, Santiago L T, Uylangco C V, Cross J H.Evaluation of sodium polyanethol sulfonate as a blood culture additivefor recovery of Salmonella typhi and Salmonella paratyphi A. J ClinMicrobiol 1983; 18:380-3

[0238] 84. Farooqui B J, Khursid M, Ashfaq M K, Khan M A. Comparativeyield of Salmonella typhi from blood and bone marrow cultures inpatients with fever of unknown origin. J Clin Pathol 1991; 44:258-9.

[0239] 85. Bystricky S, Szu S C. O-acetylation affects the bindingproperties of the carboxyl groups on the Vi bacterial polysaccharide.Biophy Chem 1994; 51:1-7.

1-10. (canceled).
 11. A method of passively immunizing a mammal againstS. typhi, comprising administering to said mammal an immunologicallysufficient amount of a first composition, the first compositioncomprising antibodies which are immunoreactive with S. typhi Vipolysaccharide, said antibodies being obtained from a human afteradministration to said human of a second composition comprising aconjugate molecule comprising the S. typhi Vi polysaccharide covalentlybound through a carboxylic acid dihydrazide linker to Pseudomonasaeruginosa recombinant exoprotein A. 12-15. (canceled).
 16. The methodof claim 11, wherein the S. typhi Vi polysaccharide comprises anN-acetyl group.
 17. The method of claim 11, wherein the S. typhi Vipolysaccharide is covalently bound to the Pseudomonas aeruginosarecombinant exoprotein A by means of an adipic acid dihydrazide linker.18. The method of claim 11, wherein the first composition isadministered to the mammal at a dosage of about 1 mg/kg to about 10mg/kg body weight of the mammal.
 19. The method of claim 11, wherein themammal is a human.
 20. The method of claim 19, wherein the human is a 2to 4 year old.
 21. The method of claim 19, wherein the human is a 5 to14 year old.
 22. A composition comprising antibodies which areimmunoreactive with S. typhi Vi polysaccharide, said antibodies beingobtained from a human after administration to said human of acomposition comprising a conjugate molecule comprising the S. typhi Vipolysaccharide covalently bound through a carboxylic acid dihydrazidelinker to Pseudomonas aeruginosa recombinant exoprotein A.
 23. Thecomposition of claim 22, further comprising antibodies which areimmunoreactive with ETA.
 24. The composition of claim 22, wherein thecomposition is chosen from the group consisting of plasma, serum, andgamma globulin fraction.
 25. The composition of claim 23, wherein thecomposition is chosen from the group consisting of plasma, serum, andgamma globulin fraction.
 26. An antibody which is immunoreactive with S.typhi Vi polysaccharide which is obtained from a human, afteradministration to said human of a composition comprising a conjugatemolecule comprising the S. typhi Vi polysaccharide covalently boundthrough a carboxylic acid dihydrazide linker to Pseudomonas aeruginosarecombinant exoprotein A.